1. Field of the Invention
The present invention relates to a test substance measurement kit and a test substance measurement method for measuring a test substance based on an antigen-antibody reaction using an insoluble carrier.
2. Description of the Related Art
A fluorescence detection method is widely used as a high-sensitive and easy measurement method for determining the amounts of protein, enzymes, inorganic compounds and the like contained in a test substance. The fluorescence detection method is a method of confirming the presence of a test substance by detecting the fluorescence produced when a test sample considered to contain the test substance, which emits the fluorescence by being excited by light having a specific wavelength, is irradiated with excitation light having the specific wavelength. When the test substance is not a fluorescent material, a method of confirming the presence of a test substance by bringing a substance, which is labeled with a fluorescent dye and specifically binds to the test substance, into contact with a test sample and by then detecting the fluorescence in the same manner as described above is also widely used.
In such a fluorescence detection method, a method using the effect of electric field enhancement by plasmon resonance to improve detection sensitivity has been known. In this method, a sensor chip provided with a metal film in a predetermined region on a transparent support is provided in order to generate plasmon resonance. Excitation light is made to incident at an angle equal to or larger than a total reflection angle from a surface of the support opposite a surface on which the metal film is formed to an interface between the support and the metal film. Due to the irradiation with the excitation light, surface plasmon is generated on the metal film, the intensity of fluorescence is increased due to an electric field enhancement action occurring by the generation of the surface plasmon, and a signal-to-noise ratio (S/N ratio) is improved. In the fluorescence detection method with the surface plasmon excitation (hereinafter, referred to as “SPF method”), an increase in signal intensity by approximately ten times is obtained compared to a fluorescence detection method with vertical illumination excitation, and thus the measurement can be performed with high sensitivity.
As an example of the SPF method, an example using a dielectric plate having a measurement region and a reference measurement region formed thereon is described in Patent Document 1. The measurement region and the reference measurement region are constituted by metal films having different thicknesses. A substance which specifically binds to a test substance is fixed to the metal film in the measurement region, and no substance is fixed to the metal film in the reference measurement region.
An immunodiagnosis system using a conventional SPF method employs a method of increasing measurement accuracy by correcting a signal value of a measurement region which reacts with a test substance with a signal value of a correction region proportional to the amount of a test sample in order to reduce errors in the measurement and a variation in the amount of a reagent contained in the product.
For example, Patent Document 2 discloses a technique including: preparing a solution in which fluorescent particles in which a first binding substance which can bind to a test substance and a third binding substance which can bind to a second binding substance having bindability to the first binding substance and does not bind to the test substance are bound to each other are mixed in a test sample; and pouring the solution into a channel in which a measurement region to which a fourth binding substance which does not bind to the first binding substance and the third binding substance, but binds to the test substance is fixed, and a correction region to which the second binding substance is fixed are formed to correct a signal detected in the measurement region using a signal detected in the correction region, thereby reducing a difference in temperature dependency between a signal value of the correction region and a signal value of the measurement region.
Patent Document 3 discloses an example in which in an analysis device in which in a sensor chip using surface plasmon resonance, an error in a detection signal due to the inclination of the sensor chip is corrected to make an accurate concentration distribution, metal films constituting a correction region and a measurement region have different thicknesses. Specifically, a sensor chip in which the metal film in the correction region has a smaller thickness than that in the measurement region and a substance which binds to a test substance is fixed only to the measurement region is used.
As a phenomenon caused by a test sample, a problem in which a positive test sample which also reacts with a negative test sample containing no test substance exists and a false positive is shown has been conventionally recognized.
Patent Document 4 describes a technique of reducing a false positive using ultrafine particles having a size of 0.2 μm or less in order to prevent a non-specific immune reaction of sensitizing particles having a size of 0.3 μm to 2.0 μm in an immunological measurement method, particularly, in an immunological measurement method using aggregation.
Patent Document 5 describes a method of detecting a test substance through an immunoagglutination reaction using sensitizing particles having a size of 0.4 μm) or greater, in which insoluble carrier particles having a size of 0.01 μm to 0.5 μm are used as particles to be used in blocking.
Patent Document 6 describes a method of adding, to particles which are smaller than particles which react specifically, a material to which an antibody or antigen which does not immunologically react with a test substance is fixed, in order to suppress a non-specific reaction.
Patent Document 7 describes a non-specific reaction inhibitor which is used in immunoassay using immunoassay particles in which an antibody or antigen immunologically reacting with a test substance is supported on a carrier having an average particle diameter of 0.05 μm to 0.5 μm, and which includes an insoluble carrier supporting an antibody or antigen which does not immunologically react with the test substance in the presence of an organic solvent, in which the average particle diameter of the insoluble carrier is smaller than that of the carrier.
Patent Document 8 describes a detection method of distinguishing between a specific binding reaction and a non-specific binding reaction between various biomolecules, in which the influence of the non-specific reaction is suppressed with particles having an outer diameter of 1 μm or less.